Our goal is to understand the molecular and biochemical processes that underlie birth defects. Although individually rare, genetic syndromes and malformations have a large impact on childhood morbidity and mortality. We are studying both a dysmorphic mouse and a human malformation syndrome. Our first model system is a genetically altered mouse that has forebrain malformations, anophthalmia and severe anemia. These malformations are due to inactivation of a gene known as Lhx2. We produced this mutation by gene targeting in embryonic stem cells. Lhx2 encodes a LIM homeobox protein that regulates the expression of other genes during development. Currently we are determining what genes are regulated by Lhx2. Using differential display PCR, we have isolated 25 candidate genes that may be regulated, directly or indirectly, by Lhx2. Sequence analysis of these clones showed that only seven of these genes were previously described genes. Several techniques including Northern Blot analysis, Ribonuclease Protection assay, and in situ hybridization analysis are being used to confirm the differential expression of these genes. Initial data indicate that two of the previously described genes appear to be differentially expressed. We have also cloned and characterized a novel LIM homeobox gene that is closely related to Lhx2. This gene is known as Lhx7. In situ expression studies have shown that Lhx7 is expressed in Cajal-Retzius neurons of the developing brain and in the developing limb. In collaboration with the Laboratory of Mammalian Genes and Development, we are producing a mouse strain in which Lhx7 does not function. This gene has been successfully mutated in embryonic stem cells and chimeric animals are now being produced. We are awaiting germline transmission of this mutation prior to proceeding with analysis of both Lhx7 mutant mice and Lhx2/Lhx7 compound mutant mice. Our laboratory is also studying the Smith-Lemli-Opitz syndrome (SLOS). This is a human malformation syndrome characterized by a characteristic facial appearance, mental retardation, hypotonia, poor growth, decreased life span and variable structural anomalies of the heart, lungs, brain, gastrointestinal tract, limbs, genitalia and kidneys. Biochemically patients with SLOS have a defect in cholesterol biosynthesis. Why these children have such a variety of malformations is not known. Initial studies with fibroblasts derived from patients with SLOS show abnormal staining with filipin. Filipin is a fluorescent dye that binds to cholesterol and to 7-dehydrocholesterol. We are currently optimizing and characterizing this staining. Our plan is to utilize this abnormal staining to test for genetic complementation between SLOS cell lines obtained from different patients.